Bacteria of the genus Chlamydia are significant pathogens of animals and man. The diseases caused by Chlamydia spp. in man include pneumonitis, endocarditis, polyarthritis, blindness, and a wide range of sexually transmitted diseases including cervicitis, salpingitis, pelvic inflammatory disease, and infertility in females; and non-gonococcal urethritis and acute epididymitis in males. Despite many years of effort, the Chlamydia remain intractable to genetic analysis due to their obligate intracellular lifestyle and complex developmental cycle. No one has been able to introduce foreign DNA into this organism and achieve stable inheritance of the expression of the foreign genes. Few attempts at isolation of Chlamydia mutants have been reported. Even cloning of Chlamydia genes by complementation has been problematic due to the absence of, or poor expression of cloned Chlamydia genes in Escherichia coli. Our long term goal is to apply the power of genetics to study the pathogenic mechanisms of Chlamydia. The goal of this proposal is to develop genetic tools for the analysis of Chlamydia biology and pathogenesis and to use these tools to address specific problems of Chlamydia pathogenesis. The Specific aims are to: 1) design an efficient, reproducible method for introduction, expression, and stable maintenance of foreign DNA in Chlamydia; 2) design genetic tools for mutagenesis and selection of mutant phenotypes of Chlamydia ; 3) clone Chlamydia genes by functional complementation; and, 4) develop a system for gene replacement in Chlamydia. Each of these aims will include development of a genetic tool, demonstration of the tools effectiveness, and application of the tool to a fundamental question of Chlamydia biology. Success in achieving these goals will have a significant impact on Chlamydia research by making new tools for genetic analysis of Chlamydia available. Rapid advances in our understanding of Chlamydia pathogenesis and biology as well as the ability to construct Chlamydia mutants for vaccine development will be made possible by these new techniques.